腸病毒71型內轉譯子及病毒蛋白質的研究

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賴雅璿(Ya-shiuan Lai) 查詢紙本館藏

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生命科學系

論文名稱

腸病毒71型內轉譯子及病毒蛋白質的研究
(Study of EV71 IRES and viral proteins)

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摘要(中)

人類的腸病毒71型 (EV71)會造成很多手足口方面的疾病並伴隨有中樞神經系統症狀。它屬於人類病毒Picornaviridae科中的Enterovirus屬。腸病毒71型完整序列大約7450個核甘酸，而在5’端的不轉譯區包含744個核甘酸，具有內轉譯子(Internal ribosome entry site; IRES)的活性，IRES可以直接幫助辨認核糖體且與之鍵結，繼而起始轉譯，此方式並不需要靠一般cap結構的幫助，故為cap-independent translation。腸病毒71型的基因體可以利用IRES轉譯產生一條約2194個胺基酸的多蛋白胜月太鏈，隨後經由病毒蛋白水解酶的切割轉趨成熟，形成11個病毒蛋白。根據前人的文獻，在酵母菌Saccharomyces cerevisiae中有一個小片段的RNA (Inhibitory RNA; IRNA)，可以選擇性的去抑制小兒痲痺病毒以及C型肝炎病毒的IRES轉譯作用，其中最主要的機制是IRNA會和細胞內某些蛋白質因子互相結合，而這些蛋白質因子恰巧為IRES進行轉譯所必需的。在本篇論文中，我們想要探究腸病毒的IRES在酵母菌裡是否可以作用，能夠在雙效表現系統中起始報告基因蛋白質的合成。而最後由無啟動子 (promoterless)的實驗，我們發現其實腸病毒71型的IRES之所以能夠起始報告基因蛋白質形成，是來自於它有潛在啟動子 (cryptic promoter)的能力。
在本篇論文的第二部份，我們也想利用酵母菌雙雜交系統去研究腸病毒71型的11個病毒蛋白在執行功能時彼此間的交互作用。發現病毒蛋白3A和2B會和自己以同複合體作用以及3A分別和2B及2C形成異複合體，而2A本身可能具有轉錄活性，除此之外，透過對酵母菌的毒性測試，只有2A會抑制酵母菌的正常生長。

摘要(英)

Human enterovirus 71 (EV71) has been implicated in numerous epidemics of hand, foot, and mouth disease (HFMD). It belongs to the Enterovirus genus of the Picornaviridae family. The 5’ untranslated region (5’ UTR) of EV71 contains 744 nucleotides and functions as internal ribosome entry site (IRES). By directly recruiting ribosomes to the IRES (cap-independent translation), EV71 genome encodes a single polypeptide chain of 2194 amino acids which is later processed to yield eleven protein products. Previous studies have shown that a small yeast RNA, known as inhibitor RNA (IRNA), can block poliovirus and HCV IRES-mediated translation by binding the host protein factors which are necessary for IRES function. In our research, we examined the ability of the 5’UTR of EV71 to play as an IRES in yeast and thus to direct the synthesis of a reporter gene in a bicistronic vector. Unfortunately, EV71 IRES in promoterless vector can still trigger the expression of the reporter gene, suggesting that EV71 IRES has a cryptic promoter activity in yeast.
In the second part of this study, we further examined if there are interactions between these eleven viral proteins by yeast two hybrid system. In our study, viral protein 3A and 2B will form homomultimer respectively in order to participate in the interaction. Still, 3A can form heteromultimer with 2B and 2C. In addition, we found that only 2A inhibits the normal growth of yeast by toxicity test and that 2A may have certain transcription activation activity.

關鍵字(中)

★ 酵母菌雙雜交系統
★ 內轉譯子
★ 腸病毒

關鍵字(英)

★ yeast two hybrid
★ IRES
★ EV71

論文目次

中文摘要 i
英文摘要 ii
誌謝 iii
目錄 iv
圖目錄 vii
表目錄 viii
符號說明 ix
第一章緒論 1
1.1. 腸病毒71型(Enterovirus 71; EV71)的簡介 1
1.2.真核細胞的兩種轉譯方式 1
1.3.IRES (Internal Ribosome Entry Site)的結構特性及分類2
1.4. 病毒的IRES在酵母菌中的作用 3
1.5.腸病毒71型十一個蛋白質的功能介紹 4
1.6.研究目的 5
第二章材料與方法 7
2.1.使用之菌株、載體及培養基 7
2.2.製備少量質體DNA（Birnboim et al., 1980） 7
2.3.鹼性去磷酸酵素（BAP）處理載體 8
2.4.接和反應（ligation） 9
2.5.大腸桿菌勝任細胞的製備與轉型作用 9
2.5.1.大腸桿菌勝任細胞的製備 10
2.6.酵母菌勝任細胞的製備與轉型作用 11
2.6.1.酵母菌勝任細胞的製備 11
2.6.2.酵母菌勝任細胞的轉型作用 12
2.7.質體之選殖 12
2.8.點突變 (Site-directed Mutagenesis) 14
2.9.蛋白質製備 (Protein preparation) 14
2.10.SDS-PAGE之蛋白質分子量分析 15
2.11.西方氏點墨法 (Western Blotting) 16
2.12.偵測Luciferase表現量 17
2.13.酵母菌雙雜交系統 (Yeast Two-Hybrid System) 18
第三章結果 20
3.1. Renilla luciferase基因可在酵母菌中當作報導基因 20
3.2.腸病毒71型的IRES可能在酵母菌中具有功能 20
3.3.酵母菌中腸病毒71型不同刪除IRES的活性測試 21
3.4.確認腸病毒71型IRES為第一型 (Type I)的IRES且是利用掃描模型起始轉譯機制 21
3.5.酵母菌中的IRNA不會抑制腸病毒71型的IRES作用 22
3.6.融合包含起始密碼的Renilla luciferase基因和上述實驗結果一致 23
3.7.腸病毒71型的IRES cDNA具有潛在啟動子 (Cryptic promoter )的能力 24
3.8.利用酵母菌雙雜交系統 (Yeast two-hybrid system)偵測腸病毒71型十一個病毒蛋白的交互作用 26
3.9.腸病毒71型中只有2A蛋白質對酵母菌INVSc1具有毒性 (Toxicity) 27
第四章討論 28
4.1.腸病毒71型IRES (1-747 bp)活性探討 28
4.2.腸病毒71型IRES利用掃描模型起始轉譯機制 29
4.3. 酵母菌中IRNA對腸病毒71型、心肌炎病毒、C型肝炎病毒的IRES作用沒有影響 29
4.4.哺乳細胞中為探討腸病毒71型真正IRES活性相關研究的模型30
4.5.腸病毒71型病毒蛋白的交互作用 30
4.6.腸病毒71型2A病毒蛋白對酵母菌INVSc1有毒性 31
第五章參考文獻 32

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指導教授

王健家(Chien-chia Wang)

審核日期

2007-7-16

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